Methods for generating pH/ionic concentration gradient near electrode surfaces for modulating biomolecular interactions

ABSTRACT

Methods of modulating the binding interactions between a (biomolecular) probe or detection agent and an analyte of interest from a biological sample in a biosensor having a multisite array of test sites. In particular, the methods modulate the pH or ionic concentration gradient near the electrodes in such biosensor. The methods of modulating the binding interactions provide a biosensor and analytic methods for more accurately measuring an analyte of interest in a biological sample.

FIELD OF THE INVENTION

The invention relates to a diagnostic method for biomolecules using abiosensor and methods of improving such biosensor. Moreover, theinvention relates to a method for generating a pH concentration gradientnear electrode surfaces for modulating biomolecular interactions in suchbiosensor.

BACKGROUND INFORMATION

Recently there has been an increased interest in predictive,preventative, and particularly personalized medicine which requiresdiagnostic tests with higher fidelity, e.g., sensitivity andspecificity. Multiplexed measurement platforms, e.g., protein arrayscurrently used in research, are among the promising diagnosticstechnologies for the near future. The samples in these tests can behuman body fluids such as blood, serum, saliva, biological cells, urine,or other biomolecules but can also be consumables such as milk, babyfood, or water. Within this field there is a growing need for low-cost,multiplexed tests for biomolecules such as nucleic acids, proteins, andalso small molecules. Achieving the sensitivity and specificity neededin such tests is not without difficult challenges. Combining these testswith integrated electronics and using CMOS technology has providedsolutions to some of the challenges.

The two main limitations in a detection assay include sensitivity andcross-reactivity. Both of these factors affect the minimum detectableconcentration and therefore the diagnostic error rate. The sensitivityin such tests is generally limited by label detection accuracy,association factor of the probe-analyte pair (for example anantibody-antigen pair), and the effective density of probe molecule (forexample probe antibody) on the surface (as shown in FIG. 1). Othermolecules in the biological sample can also affect the minimumdetectable concentration by binding to the probe molecule (for examplethe primary antibody), or by physisorption of the analyte to the surfaceat the test site (as shown in FIG. 2). The detection agent (for examplea secondary antibody) may also physisorb to the surface causing anincrease in the background signal (as shown in FIG. 2). Solving thecross-reactivity and background problem can take a significant amount oftime in the assay development of a new test and increases the cost andcomplexity of the overall test. The assay is typically optimized byfinding the best reagents and conditions and also by manufacturing themost specific probe molecule (for example antibody). This results in along development time, the infeasibility of tests in some cases, and ahigher manufacturing cost. For example a typical development of an ELISAassay requires several scientists working for more than a year findingthe correct antibody as part of the assay development. Cross-reactivityof the proteins may be the source of the failure of such an effort.

A biosensor providing a multiple site testing platform was thought toprovide a solution to some of the above described limitations in assaydevelopment. US Published Patent Application US 2011/0091870 describessuch biosensor having multiple sites that could be subjected todifferent reaction conditions to modulate the binding of thebiomolecular analyte (for example proteins) to the probe molecule. Forexample the signal detected in a biosensor having four sites also canhave several components, e.g. four. These four terms may correspond tothe concentration of the biomarker of interest, concentration ofinterfering analytes in the sample that bind non-specifically to primaryantibody (probe molecule) sites and prevent the biomarker to bind,concentration of interfering analytes in the sample that form a sandwichand produce wrong signal, and finally the concentration of interferinganalytes in the sample that physisorb to the surface and produce wrongsignal. Each term is also proportional to a binding efficiency factor,α_(ij), which is a function of the molecule affinities and other assayconditions, e.g., mass transport. By controlling the condition at eachsite separately, different sites will have different efficiency factors.Accurate measurement of the signal at each site will result in multipleequations and multiple unknowns for example,

$\left\{ {\begin{matrix}{S_{1} = {{\alpha_{11}C_{an}} - {\alpha_{12}C_{j\; 1}} + {\alpha_{13}C_{j\; 2}} + {\alpha_{14}C_{j\; 3}}}} \\{S_{2} = {{\alpha_{21}C_{an}} - {\alpha_{22}C_{j\; 1}} + {\alpha_{23}C_{j\; 2}} + {\alpha_{24}C_{j\; 3}}}} \\{S_{3} = {{\alpha_{31}C_{an}} - {\alpha_{32}C_{j\; 1}} + {\alpha_{33}C_{j\; 2}} + {\alpha_{34}C_{j\; 3}}}} \\{S_{4} = {{\alpha_{41}C_{an}} - {\alpha_{42}C_{j\; 1}} + {\alpha_{43}C_{j\; 2}} + {\alpha_{44}C_{j\; 3}}}}\end{matrix}C_{an}} \right.$where C_(an) corresponds to the targeted biomolecular analyteconcentration and C_(j1), C_(j2), C_(j3) correspond to the totalconcentration of molecules which result in different terms in backgroundsignal.

Accurate and precise control of the assay conditions at different sitesto generate large changes in the binding efficiency factors is importantin the performance of such biosensor as a detection system for abiomolecular analyte of interest. Thus, there is a need for methods thatcan be readily integrated with a CMOS, electrode array, or TFT basedsetup to generate large change in binding efficiencies between testsites in a biosensor having an array of multiple test sites.

SUMMARY OF THE INVENTION

Herein are provided such methods that can be integrated with for examplea CMOS, electrode array, or TFT based biosensor to generate largechanges in binding efficiencies between test sites in the biosensorhaving an array of multiple test sites. In particular, the currentapplication provides methods to modulate the pH or ionic concentrationnear electrode surfaces of such biosensors in order to modulate thebiomolecular interactions between a probe biomolecule and a biomolecularanalyte of interest.

In one embodiment there is provided a method of modulating the pH orionic concentration in a biosensor, the method comprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecule interface layer having one or moreimmobilized probes thereon;

b) adding an electrochemically active agent, an enzyme, an enzymesubstrate, a buffer inhibitor, or a combination thereof to the aqueoussolution; and

c) reacting the electrochemically active agent, the enzyme, the enzymesubstrate, or a combination thereof in the aqueous solution to produceH⁺ ion or OH⁻ ions, or increasing the diffusion of H⁺ ions or OH⁻ ionswith the buffer inhibitor, or inhibiting the interaction between H⁺ ionsor OH⁻ ions and buffering salts with the buffer inhibitor.

In another embodiment there is provided a method of modulating the pH orionic concentration in a biosensor, the method comprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecule interface layer having one or moreimmobilized probes;

b) adding an electrochemically active agent to the aqueous solution; and

c) oxidizing or reducing the electrochemically active agent.

In another embodiment there is provided a method of modulating the pH orionic concentration in a biosensor, the method comprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecule interface layer having one or moreimmobilized probes and one or more immobilized enzymes thereon;

b) adding an enzyme substrate to the aqueous solution; and

c) enzymatically oxidizing or reducing the enzyme substrate.

In yet another embodiment there is provided a method of modulating thepH or ionic concentration in a biosensor, the method comprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising an electromagnet in anaqueous solution and a biomolecule interface layer having one or moreimmobilized probes;

b) adding one or more enzymes immobilized onto magnetic micro- ornano-particles to the aqueous solution;

c) adding an enzyme substrate to the aqueous solution; and

d) enzymatically oxidizing or reducing the enzyme substrate.

In another embodiment there is provided a method of modulating the pH orionic concentration in a biosensor, the method comprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecule interface layer having one or moreimmobilized probes;

b) adding an enzyme to the aqueous solution; and

c) reacting the enzyme at the electrode surface.

In another embodiment there is provided a method of modulating the pH orionic concentration in a biosensor, the method comprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecule interface layer having one or moreimmobilized probes;

b) adding a buffer inhibitor to the aqueous solution; and

c) inhibiting the diffusion of H⁺ ions or OH⁻ ions or the interactionbetween H⁺ ions or OH⁻ ions and buffering salts.

In yet another embodiment there is provided a biosensor for use indetecting a biomolecule analyte comprising a multisite array of testsites in which the conditions for interacting with the biomoleculeanalyte can be varied independently, each test site comprising:

a) a support in an aqueous environment;

b) one or more electrodes; and

c) a biomolecule interface layer having one or more immobilized probesand one or more immobilized enzymes.

In another embodiment there is provided a method for detecting abiomolecule analyte in a biological sample, the method comprises:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution comprising a water-miscible organic co-solvent, e.g.,acetonitrile, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), andN,N-dimethyl acetamide (DMAc), to facilitate the dissolution of anelectrochemical active agent, the support comprising one or moreelectrodes or an electromagnet, and a biomolecule interface layer havingone or more immobilized probes thereon;

b) adding in each test site an electrochemically active agent, anenzyme, an enzyme substrate, a buffer inhibitor, or a combinationthereof to the aqueous solution;

c) reacting the electrochemically active agent, the enzyme, the enzymesubstrate, or a combination thereof in the aqueous solution to produceH⁺ ion or OH⁻ ions, or increasing the diffusion of H⁺ ions or OH⁻ ionswith the buffer inhibitor, or the inhibiting the interaction between H⁺ions or OH⁻ ions and buffering salts with the buffer inhibitor;

d) adding a biological sample to each test site; and

e) detecting the biomolecule analyte in each test site,

wherein the amounts added in step b) and the reaction in step c) arevaried between test sites in a subset array of test sites in order toobtain sets of test sites in which the pH or ionic concentration nearthe electrode surfaces in the test sites varies.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Illustration of the steps of a typical and well known ELISAassay: a) Sample introduced to immobilized primary antibody on a blockedsurface and incubated, b) Sample washed, and c) labeled secondaryantibody is added. The number of labels is proportional to theconcentration of target antigen.

FIG. 2: Illustration of the undesired cross-reactivity. Molecules otherthan the antigen of interest (diamond) can bind to primary antibody orthe surface and either create incorrect signal or prevent the antigen informing a sandwich.

FIG. 3: Illustration of the multisite sensor and the components in thedetected signal. The two schematics on the bottom correspond to two ofthe sites.

FIG. 4: Illustration of the composition of a sensor test site in amultisite sensor.

FIG. 5: Schematic of the pH change on an electrode surface usingelectrochemical method.

FIG. 6: Illustration of pH change by enzymatic reactions when they arebrought close to the protein surface using magnetic micro/nanoparticles.The micro/nano cavity helps in localizing the pH change.

FIG. 7: A) Cyclic voltammograms of Indium Tin Oxide (ITO) electrodes inPBS only. The region where pH change can occur is where there is oxygenevolution more than 1V in respect to Ag/AgCl reference electrode. B):Cyclic voltammetric study of the oxidation of Ascorbic acid test in ITOelectrodes. Current increase around 0.25V indicates the start ofoxidation at ITO electrodes.

FIG. 8: A) Application of 1V on the ITO-PEG surface in Phosphate buffer.Impedance changes before and after application of 1V indicates thechanges or removal of PEG from electrode. B): Oxidation of ascorbic acidat 0.5V and 0.75V at ITO-PEG surface. No change in impedance duringascorbic acid oxidation indicates PEG layers do not undergo any change.

DETAILED DESCRIPTION

In order to vary the pH or ionic concentration gradient in a multisitearray of test sites in a biosensor there is provided a method ofmodulating the pH or ionic concentration in a biosensor, the methodcomprising:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecule interface layer having one or moreimmobilized probes thereon;

b) adding an electrochemically active agent, an enzyme, an enzymesubstrate, a buffer inhibitor, or a combination thereof to the aqueoussolution; and

c) reacting the electrochemically active agent, the enzyme, the enzymesubstrate, or a combination thereof in the aqueous solution to produceH⁺ ion or OH⁻ ions, or increasing the diffusion of H+ ions or OH− ionswith the buffering agent or inhibiting the interaction between H⁺ ionsor OH⁻ ions and buffering salts with the buffer inhibitor.

In the above described method a local pH or ionic concentration gradientcan be obtained in the various test sites in a multisite arraybiosensor. The variation of the local pH and/or ionic concentrationgradient at the electrode, and in particular in the vicinity of the(biomolecular) probe in a biomolecular interface layer, over subsets ofthe multisite array of the biosensor, allows for modulating the bindingefficiency of the (biomolecular) probe and an analyte to be tested froma biological sample. The analyte of interest, when bound to the(biomolecular) probe, can be then detected using a detection agent, suchas for example a labeled secondary antibody. The modulation of bindingefficiencies in a subset of a multisite array provides a method for theaccurate determination of such analyte of interest.

The biosensor preferably comprises a multisite array of test sites asfor example is described in US 2011/0091870. Such multisite arraypreferably includes a number of different subarrays/subsets of testsites. Each test sites represents a site for performing an analysis of a(biomolecular) analyte from a biological sample through the detection ofthe (biomolecular) analyte using a (biomolecular) probe. The analyticalconditions in each test site in each of the subarrays/subsets may bevaried to obtain a collection of varied signals that will result inmultiple equations and multiple unknowns from which the concentration ofthe (biomolecular) analyte can be determined in order to obtain anaccurate measurement of the (biomolecular) analyte.

The multiple unknowns in the obtained varied signals each includes aterm that is proportional to a binding efficiency factor, α_(ij), andthe concentrations of the various molecules in the biological samplebinding that are detected at the test site. The multiple equations withmultiple unknowns may be represented for example as follows,

$\left\{ {\begin{matrix}{S_{1} = {{\alpha_{11}C_{an}} - {\alpha_{12}C_{j\; 1}} + {\alpha_{13}C_{j\; 2}} + {\alpha_{14}C_{j\; 3}}}} \\{S_{2} = {{\alpha_{21}C_{an}} - {\alpha_{22}C_{j\; 1}} + {\alpha_{23}C_{j\; 2}} + {\alpha_{24}C_{j\; 3}}}} \\{S_{3} = {{\alpha_{31}C_{an}} - {\alpha_{32}C_{j\; 1}} + {\alpha_{33}C_{j\; 2}} + {\alpha_{34}C_{j\; 3}}}} \\{S_{4} = {{\alpha_{41}C_{an}} - {\alpha_{42}C_{j\; 1}} + {\alpha_{43}C_{j\; 2}} + {\alpha_{44}C_{j\; 3}}}}\end{matrix}C_{an}} \right.$where C_(an) corresponds to the targeted biomolecular analyteconcentration and C_(j1), C_(j2), C_(j3) correspond to the totalconcentration of molecules which result in different terms in backgroundsignal, from which collection of multiple equations the concentration ofthe targeted biomolecular analyte can be determined.

The number of subarrays/subsets, as well as the number of test siteswithin each subarray/subset may be varied, as needed to obtain suchaccurate measurement of the analyte. Some of these analytical conditionsinclude parameters such as for example temperature, shear stress, andpressure. For example the temperature of the aqueous solution in whichthe biomolecular probe and analyte of interest in the biological sampleinteract can be varied using the electromagnetic heat at the test site.Another important condition for the interaction between the biomolecularprobe and the analyte of interest is the pH or ionic concentration. Themethod described herein modulates this pH or ionic concentration in thelocal environment of the biomolecular probe in order to affect thebinding efficiency in the vicinity of the biomolecular probe.

Each test site in the subarray/subset of the multiple site arraycomprises a support onto which one or more electrodes are placed andonto which solid surface the biomolecular probe(s) are immobilized orbound (as shown in FIGS. 3 and 4). This immobilization of biomolecularprobes to a solid surface or support assists in reducing the amount ofprobe needed for the analytical method and also localizes the detectionarea to make accurate measurements. The biomolecular probes aretherefore attached to solid surfaces of the support and/or electrodessuch as those of silicon, glass, metal and semiconductor materials (asshown in FIG. 4).

The biomolecular probe is attached or immobilized onto the supportand/or electrode(s) within a biomolecular interface layer (as shown inFIG. 4). The biomolecular layer includes a layer of immobilizedpolymers, preferably a silane immobilized polyethylene glycol (PEG).Surface-immobilized polyethylene glycol (PEG) can be used to preventnon-specific adsorption of biomolecular analytes onto surfaces. At leasta portion of the surface-immobilized PEG can comprise terminalfunctional groups such as N-hydroxysuccinimide (NHS) ester, maleimide,alkynes, azides, streptavidin or biotin that are capable of conjugating.The biomolecular probe may be immobilized by conjugating with thesurface-immobilized PEG. It is important that the method used to changethe pH does not impair the covalent binding of for example the PEG ontothe surface of a solid support, or the linker that conjugated thebiomolecular probe to the PEG (as shown in FIG. 5). The method ofmodulating the pH or ionic concentration as described herein can protectthese surface chemistries, while affecting a pH/ionic concentrationchange in the environment of the biomolecular probe.

A suitable biomolecular probe can be a carbohydrate, a protein, aglycoprotein, a glycoconjugate, a nucleic acid, a cell, or a ligand forwhich the analyte of interest has a specific affinity. Such probe canfor example be an antibody, an antibody fragment, a peptide, anoligonucleotide, a DNA oligonucleotide, a RNA oligonucleotide, a lipid,a lectin that binds with glycoproteins and glycolipids on the surface ofa cell, a sugar, an agonist, or antagonist. In a specific example, thebiomolecular probe is a protein antibody which interacts with an antigenthat is present for example in a biological sample, the antigen being abiomolecular analyte of interest.

In the analytical method described herein the analyte of interest in abiological sample can be for example a protein, such as an antigen orenzyme or peptide, a whole cell, components of a cell membrane, anucleic acid, such as DNA or RNA, or a DNA oligonucleotide, or a RNAoligonucleotide.

The electrodes can be any electrode suitable in a biosensor for exampleindium tin oxide (ITO), gold, or silver electrodes. In a preferredembodiment the electrodes in the biosensor in the method describedherein are indium tin oxide (ITO) electrodes.

This analytical method using a biosensor for detecting a biomoleculeanalyte in a biological sample according to one embodiment is a methodwhich comprises the steps of:

a) providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecule interface layer having one or moreimmobilized detection agents thereon;

b) adding in each test site an electrochemically active agent, anenzyme, an enzyme substrate, a buffer inhibitor, or a combinationthereof to the aqueous solution;

c) reacting the electrochemically active agent, the enzyme, the enzymesubstrate, or combination thereof in the aqueous solution to produce H⁺ion or OH⁻ ions, or increasing the diffusion of H⁺ ions or OH⁻ ions withthe buffer inhibitor, or inhibiting the interaction between H⁺ ions orOH⁻ ions and buffering salts with the buffer inhibitor;

d) adding a biological sample to each test site; and

e) detecting the biomolecule analyte in each test site,

wherein the amounts added in step b) and the reaction in step c) arevaried between test sites in a subset array of test sites in order toobtain sets of test sites in which the pH or ionic concentration nearthe electrode surfaces in the test sites varies. The aqueous solutioncomprises a water-miscible organic co-solvent, e.g., acetonitrile,dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), and N,N-dimethylacetamide (DMAc), to facilitate the dissolution of an electrochemicalactive agent. The amount of water-miscible organic solvent can rangefrom 0.1 to 80% v/v, preferably from 0.1 to 10% v/v, most preferablyfrom 1.0 to 5.0% v/v in respect to water in the aqueous solution. Theanalytical method hereby obtains in each subset of test sites a pH orionic concentration gradient over the test sites in the subset in thevicinity of the biomolecular probe. The binding efficiencies of theanalyte and any other molecule in the biological sample is therebydifferently affected in each series of test sites in each subset.

The biomolecular analyte can be detected using any suitable detectionmethod. Known detection methods of such analyte include luminescence,fluorescence, colorimetric methods, electrochemical methods, impedancemeasurements, or magnetic induction measurements. In various of suchmethods the analyte binds to the immobilized biomolecular probe and adetection agent such as for example a secondary labeled probe thatspecifically binds to the analyte, bound to the immobilized probe, isintroduced. This detection agent or secondary labeled probe gives riseto a detectable signal such as for example luminescence or fluorescence(as shown in FIGS. 5 and 6).

In such analytical method the pH of the solution surrounding theimmobilize biomolecular probe has been known to influence thebinding/activity between the probe and the analyte to a great extent.Concentration of other ions on surrounding proteins can also heavilyinfluence the binding/activity. Herein are provided methods to modulatethe pH and/or ionic concentration in the vicinity of the biomolecularprobe immobilized close to a surface. The modulation of the pH nearthese solid surfaces also affect the non-specific interactions of theanalyte to other molecules than the biomolecular probe and theinteractions of other molecules in the solution of the biological samplewith the biomolecular probe or analyte. The modulation of pH or ionicconcentration however should not impair any of the surface chemistries,such as those that immobilize the biomolecular probe to its solidsupport in a test site of a multisite array in the biosensor. The methodof modulating the pH or ionic concentration as described herein canprotect these surface chemistries, while affecting a pH/ionicconcentration change in the environment of the biomolecular probe.

Surface chemistry compatibility is an important consideration thatshould be noted when the methods described herein are practiced. pHchange is caused by changes in hydrogen ion or hydroxyl ionconcentrations. A variety of chemical reactions taken place atelectrode-liquid, electrode-cross linker, cross linker-protein, andprotein-protein interfaces as shown in FIG. 5 can also become ahindrance to pH changes happening near the solid surfaces to reach theproteins on top of them. They can simply act as diffusion barriers forthe ions and hinder the pH changes around the biomolecular probes andanalytes. These methods of modulating the pH or ionic concentrationdescribed herein helps in maximizing the changes in hydrogen or hydroxylion concentration so that they can overcome any diffusion barrierimposed by the surface chemistry.

Another important aspect is the buffering capacity of the solution incontact with the solid interface. The buffering effect can be largeenough that the pH change at the interface would never reach thebiomolecular probes that are immobilized away from it. The distance canvary based on the biomolecular interface layer deposited on top of thesolid interface. Such biomolecular interface layer may have a thicknessof 300 nm or less, preferable between 1-150 nm, even more preferablybetween 5-100 nm. As such the distance between the solid interface andthe biomolecular probe within the biomolecular interface layer can rangebetween 0.1-300 nm. Use of buffer inhibitors in the solution or on thesurface that extend the pH change on the electrode interface to reachthe interacting probe-analyte pair may contribute to modulating the pHor ionic concentration in the vicinity of the biomolecular probe.

Following are examples of methods for modulating the pH/ionicconcentration at the solid-liquid interfaces. These include: 1) theelectrochemical generation of ions at electrode surfaces by adding anelectrochemically active species to the solution which generates ions ofinterest (e.g., H+, Mg+, OH−) upon electrochemical oxidation/reduction;2) bringing enzymes close to the site of interest, which release suchions of interest from an enzyme substrate that is reacted with theenzyme; 3) the introduction of buffer inhibitors, for example, by mixingpolymers that selectively reduce the diffusion rate of ions in thesolution (e.g., phosphate). U.S. Pat. No. 7,948,015 describes the use ofsuch inhibitors for applications in which measuring small local pHchanges is of interest (e.g., in DNA sequencing). However in the methodsof locally modulating the pH similar inhibitors can be used in order toextend the local pH changes further away from the electrode-liquidinterface; and 4) the redistribution of preexisting ions near theelectrode surface due to electrostatic forces.

In one embodiment of a method for modulating the pH or ionicconcentration in a biosensor as described herein an electrochemicallyactive agent is added to the aqueous solution at a test site in amultisite array, wherein the test site has a biomolecular interfacelayer comprising a biomolecular probe or detection agent and oxidizingor reducing the electrochemically active agent. The electrochemicallyactive agent may be added at a concentration of 1 nM to 100 mM,preferably at a concentration between 10 nM and 10 mM, more preferablyat a concentration of 100 nM and 5 mM. The electrochemically activeagent may be electro-oxidized or electro-reduced at an electrodepotential in the range of −2V to +2V (vs. Ag/AgCl reference electrode).Preferably the electrode potential is in the range of −1V to +1V, evenmore preferably the electrode potential is in the range of −0.5V to+0.5V.

Suitable electrochemically active agents include dopamine hydrochloride,ascorbic acid, phenol and derivatives, benzoquinones and derivatives,for example, 2,5-dihydroxy-1,4-benzoquinone,2,3,5,6-tetrahydroxy-1,4-benzoquinone and2,6-dichloroquinone-4-chloroimide; naphthoquinones and derivatives, forexample, hydroxy-1,4-naphthoquinone, 5,8-dihydroxy-1,4-naphthoquinone,and potassium 1,4-naphthoquinone-2-sulfonate; and 9,10-anthraquinone andderivatives, for example, sodium anthraquinone-2-carboxylate, potassium9,10-anthraquinone-2,6-disulfonate.

In another embodiment of a method for modulating the pH or ionicconcentration in a biosensor, an enzyme is immobilized in a biomoleculeinterface layer also having one or more immobilized biomolecularprobers. An enzyme substrate is then added to the aqueous solution at atest site in a multisite array, wherein the test site has thebiomolecular interface layer and enzymatically oxidizing the enzymesubstrate.

In another embodiment of a method for modulating the pH or ionicconcentration, the method comprises:

a) providing a biosensor comprising an electromagnet in an aqueoussolution and a biomolecule interface layer having one or moreimmobilized detection agents;

b) adding one or more enzymes immobilized onto magnetic micro- ornano-particles to the aqueous solution;

c) adding an enzyme substrate to the aqueous solution; and

d) enzymatically oxidizing the enzyme substrate.

Suitable enzymes for immobilization in the biomolecular interface layeror onto the magnetic micro- or nano-particles include for exampleoxidases, ureases, or dehydrogenases. Such immobilized oxidase is forexample a glucose oxidase and the enzyme substrate is glucose. Theamounts of immobilized enzyme and enzyme substrate added can be variedin the different test sites in each of the subsets of the multisitearray so as to provide a pH or ionic concentration gradient in the suchsubset of the multisite array.

Alternatively the enzyme is not immobilized onto a solid surface such asin the above methods being immobilized into a biomolecular interfacelayer or onto a magnetic micro- or nano-particle but is added to theaqueous solution in the test sites of subsets of a multisite array.Through electrolysis the enzyme undergoes a redox reaction at theelectrode surface and perturbs the local pH.

In each of these embodiments the pH or ionic concentration can befurther modulated by adding a buffer inhibitor to the aqueous solution.Such addition of a buffer inhibitor either assists in diffusing theproduced ions of interest to the location of the biomolecular probe ordetection agent or inhibits the interaction of such produced ions withbuffering salts. Alternatively, in the method of modulating the pH orionic concentration in a biosensor as described herein, the bufferinhibitor is added to the aqueous solution of the test site of subsetsof a multisite array in the absence of an electrochemical active agentor immobilized enzyme. In such embodiment the buffer inhibitor is addedto the aqueous solution, and facilitates the diffusion of H⁺ ions or OH⁻ions that are produced at the electrodes in the test site or inhibitsthe interaction between H⁺ ions or OH⁻ ions and buffering salts.

Suitable buffer inhibitors include soluble polymers selected frompoly(allylamine hydrochloride), poly(diallyldimethyl ammonium chloride),poly(vinylpyrroldone), poly(ethyleneimine), poly(vinylamine),poly(4-vinylpyridine) and tris(2-carboxyethyl)phosphine hydrochloride.The amounts of buffer inhibitor added can be varied in the differenttest sites in each of the subsets of the multisite array so as toprovide a pH or ionic concentration gradient in the such subset of themultisite array.

The following are examples which illustrate specific methods without theintention to be limiting in any manner. The examples may be modifiedwithin the scope of the description as would be understood from theprevailing knowledge.

EXAMPLES

Electrochemical Generation of H+ or OH− Ions at Electrode Surfaces

Electrode material used: The electrode material was indium tin oxide.This is a semiconducting electrode surface with very large potentialwindow in an aqueous solution.

Electro-Oxidation of Species to Produce H+ Ions

Oxidation of ascorbic acid at the electrode surfaces produced H+ ionsand changed the electrode surface pH to a more acidic state:AH₂→A+2H⁺+2e ⁻,where AH₂ is ascorbic acid (C₆H₆O₆) (as shown in FIG. 5). The electrodepotential at which it oxidizes was less than 0.5V for Indium tin oxidematerial vs Ag/AgCl reference electrode (as shown in FIG. 7). Thispotential was less than the voltages needed for the oxygen evolutionreaction in aqueous solution. Higher electrode potential (e.g >1V forITO electrodes in just phosphate buffer) can damage the PEG layer (asshown in FIG. 8). The ascorbic acid also acted as a sacrificial speciesto prevent electrochemical degradation of the surface chemistry.

Electro-Reduction of Species to Produce OH− Ions

Reduction of benzoquinone (C₆H₄O₂) into Hydroquinone (C₆H₆O₂) canproduce OH⁻ ions at −0.1VBQ+2e ⁻+2H₂O→HQ+20H⁻This reduction reaction increased the pH at the electrode interface.

In the above examples the amount of H⁺ or OH⁻ ions generated will dependon the concentration of species present in solution (nM-mM range),potential applied (−2V to +2V), type of waveform (pulse, constant,sawtooth, sinusoidal, square wave at different frequencies and dutycycles), and diffusion of the species (can be varied due to additives inthe solution). These parameters can be optimized to get different pHs atthe each of the electrode element present in the multisite biosensor.

pH Change Using Enzymatic Reactions

Enzymes such as oxidases, ureases or dehydrogenases have been known toconsume or generate hydrogen during the reaction. For example:β-d-glucose+O₂→d-glucose-δ-lactone+H₂O₂d-glucose-δ-lactone+H₂O→d-gluconate+H⁺Oxidation of glucose in the presence of glucose oxidase can produce H+ions that are used to change the pH near the proteins of interest.

Co-Immobilization of Enzymes Along with Biomolecular Probes in aBiomolecular Interface Layer

The enzymes when co-immobilized on the surface along with proteinsbrings them in close proximity so the H⁺ produced by the enzymaticreaction will lead to a localized pH change that can affect proteinbinding (for example antigen-antibody binding and non-specific binding).

Attaching the Enzymes to Magnetic Micro/Nanoparticles

Proteins are attached to micro/nanocavities of a solid surface on anelectromagnet. The enzymes are separately attached to magneticmicro/nanoparticles in the solution. Controlling the electromagnet thatis fabricated/placed underneath controls the local pH values. Then theenzymatic reaction is triggered by introducing the corresponding enzymesubstrate (as shown in FIG. 6). Alternatively electrochemically activeenzymes are used. The pH change is localized on the cavities and theprotein interactions are modulated.

What is claimed is:
 1. A method comprising: a) providing a biosensorcomprising a multisite array of test sites, each test site comprising asupport in an aqueous solution, the support comprising one or moreelectrodes or an electromagnet, and a biomolecule interface layer havingone or more immobilized probes thereon; b) adding an electrochemicallyactive agent, an enzyme, an enzyme substrate, a buffer inhibitor, or acombination thereof to the aqueous solution; c) during a test,independently varying for each of the test sites a degree of biologicalinteraction that can occur with a biomolecule analyte at the respectivetest site, to obtain a collection of varied signals, wherein the varyingincludes reacting the electrochemically active agent, the enzyme, theenzyme substrate, or combination thereof in the aqueous solution toproduce H⁺ ion or OH⁻ ions, or increasing the diffusion of H⁺ ions orOH⁻ ions with the buffer inhibitor, or inhibiting the interactionbetween H⁺ ions or OH⁻ ions and buffering salts with the bufferinhibitor; and d) analyzing the collection of varied signals to obtain ameasurement.
 2. A method of modulating the pH or ionic concentration ina biosensor, the method comprising: a) providing a biosensor comprisinga multisite array of test sites in which the conditions for interactingwith a biomolecule analyte can be varied independently, each test sitecomprising a support in an aqueous solution, the support comprising oneor more electrodes or an electromagnet, and a biomolecule interfacelayer having one or more immobilized probes thereon; b) adding anelectrochemically active agent, an enzyme, an enzyme substrate, a bufferinhibitor, or a combination thereof to the aqueous solution; and c)reacting the electrochemically active agent, the enzyme, the enzymesubstrate, or combination thereof in the aqueous solution to produce H⁺ion or OH⁻ ions, or increasing the diffusion of H⁺ ions or OH⁻ ions withthe buffer inhibitor, or inhibiting the interaction between H⁺ ions orOH⁻ ions and buffering salts with the buffer inhibitor; wherein: theaqueous solution comprises a water-miscible organic co-solvent selectedfrom the groups consisting of acetonitrile, dimethyl sulfoxide (DMSO),dimethyl formamide (DMF), N,N-dimethyl acetamide (DMAc), and mixturesthereof; the biomolecular interface layer comprises a silane immobilizedpolyethylene glycol (PEG), and at least a portion of thesurface-immobilized PEG comprises terminal functional groups selectedfrom the group consisting N-hydroxysuccinimide (NHS) ester, maleimide,alkynes, azides, streptavidin and biotin; the biosensor comprises asupport in an aqueous solution, the support comprising one or moreelectrodes, and a biomolecule interface layer having one or moreimmobilized probes and one or more immobilized enzymes, in step b) anenzyme substrate is added to the aqueous solution, and in step c) theenzyme substrate is enzymatically oxidized or reduced; or the supportcomprises an electromagnet and the biomolecule interface layer has oneor more immobilized probes, in step b) one or more enzymes immobilizedonto magnetic micro- or nano-particles are added prior to orsimultaneously with an enzyme substrate, and in step c) the enzymesubstrate is enzymatically oxidized or reduced.
 3. The method ofmodulating the pH or ionic concentration in a biosensor according toclaim 1, wherein the biomolecular interface layer having one or moreimmobilized probes thereon comprises a layer of immobilized polymers andthe probes.
 4. The method of modulating the pH or ionic concentration ina biosensor according to claim 2, wherein the biomolecular interfacelayer comprises the silane immobilized polyethylene glycol (PEG), and atleast a portion of the surface-immobilized PEG comprises the terminalfunctional groups selected from the group consistingN-hydroxysuccinimide (NHS) ester, maleimide, alkynes, azides,streptavidin and biotin.
 5. The method of modulating the pH or ionicconcentration in a biosensor according to claim 1, wherein in step b) anelectrochemically active agent is added to the aqueous solution, and instep c) the electrochemically active agent is oxidized or reduced. 6.The method of modulating the pH or ionic concentration in a biosensoraccording to claim 5, wherein the electrode potential is in the range of−1V to +1V vs Ag/AgCl.
 7. The method of modulating the pH or ionicconcentration in a biosensor according to claim 5, further comprisingadding a buffer inhibitor to the aqueous solution.
 8. The method ofmodulating the pH or ionic concentration in a biosensor according toclaim 1, wherein the electrochemically active agent is added at aconcentration of 1 nM to 100 mM.
 9. The method of modulating the pH orionic concentration in a biosensor according to claim 1, wherein theelectrochemically active agent is selected from the group consisting ofdopamine hydrochloride, ascorbic acid, phenol and derivatives thereof,benzoquinones and derivatives thereof, naphthoquinones and derivativesthereof, and 9,10-anthraquinone and derivatives thereof.
 10. The methodof modulating the pH or ionic concentration in a biosensor according toclaim 1, wherein the buffer inhibitor is a soluble polymer selected frompoly(allylamine hydrochloride), poly(diallyldimethyl ammonium chloride),poly(vinylpyrroldone), poly(ethyleneimine), poly(vinylamine),poly(4-vinylpyridine) and tris(2-carboxyethyl)phosphine hydrochloride.11. The method of modulating the pH or ionic concentration in abiosensor according to claim 1, wherein the biosensor comprises a CMOS,electrode array, or TFT based system.
 12. The method of modulating thepH or ionic concentration in a biosensor according to claim 1, whereinthe electrode is an indium tin oxide, gold or silver electrode.
 13. Themethod of modulating the pH or ionic concentration in a biosensoraccording to claim 12, wherein the protein is an antibody.
 14. Themethod of modulating the pH or ionic concentration in a biosensoraccording to claim 1, wherein the support is a glass or plastic support.15. The method of modulating the pH or ionic concentration in abiosensor according to claim 1, wherein the immobilized probe isselected from a protein, a peptide, a nucleic acid and a cell.
 16. Themethod of modulating the pH or ionic concentration in a biosensoraccording to claim 2, wherein the aqueous solution comprises thewater-miscible organic co-solvent selected from the groups consisting ofacetonitrile, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF),N,N-dimethyl acetamide (DMAc), and mixtures thereof.
 17. The method ofmodulating the pH or ionic concentration in a biosensor according toclaim 2, wherein the biosensor comprises the support in the aqueoussolution, the support comprising the one or more electrodes, and thebiomolecule interface layer having the one or more immobilized probesand one or more immobilized enzymes, in step b) the enzyme substrate isadded to the aqueous solution, and in step c) the enzyme substrate isenzymatically oxidized or reduced.
 18. The method of modulating the pHor ionic concentration in a biosensor according to claim 17, wherein theimmobilized enzyme is an oxidase, urease, or dehydrogenase.
 19. Themethod of modulating the pH or ionic concentration in a biosensoraccording to claim 17, further comprising adding a buffer inhibitor tothe aqueous solution.
 20. The method of modulating the pH or ionicconcentration in a biosensor according to claim 2, wherein the supportcomprises the electromagnet and the biomolecule interface layer has theone or more immobilized probes, in step b) the one or more enzymesimmobilized onto magnetic micro- or nano-particles are added prior to orsimultaneously with the enzyme substrate, and in step c) the enzymesubstrate is enzymatically oxidized or reduced.
 21. The method ofmodulating the pH or ionic concentration in a biosensor according toclaim 20, wherein the immobilized enzyme is an oxidase, urease, ordehydrogenase.
 22. The method of modulating the pH or ionicconcentration in a biosensor according to claim 20, further comprisingadding a buffer inhibitor to the aqueous solution.
 23. A method ofmodulating the pH or ionic concentration in a biosensor, the methodcomprising: a) providing a biosensor comprising a multisite array oftest sites in which the conditions for interacting with a biomoleculeanalyte can be varied independently, each test site comprising a supportin an aqueous solution, the support comprising one or more electrodes oran electromagnet, and a biomolecule interface layer having one or moreimmobilized probes thereon; b) adding an enzyme to the aqueous solution;and c) reacting the enzyme at the electrode surface.
 24. The method ofmodulating the pH or ionic concentration in a biosensor according toclaim 23, further comprising adding a buffer inhibitor to the aqueoussolution.
 25. A method of modulating the pH or ionic concentration in abiosensor, the method comprising: a) providing a biosensor comprising amultisite array of test sites in which the conditions for interactingwith a biomolecule analyte can be varied independently, each test sitecomprising a support in an aqueous solution, the support comprising oneor more electrodes or an electromagnet, and a biomolecule interfacelayer having one or more immobilized probes thereon; b) adding a bufferinhibitor to the aqueous solution; and c) reacting the electrochemicallyactive agent, the enzyme, the enzyme substrate, or combination thereofin the aqueous solution to produce H⁺ ion or OH⁻ ions, or increasingdiffusion of H⁺ ions or OH⁻ ions with the buffer inhibitor, orinhibiting the interaction between H⁺ ions or OH⁻ ions and bufferingsalts is inhibited with the buffer inhibitor.
 26. A method of modulatingthe pH or ionic concentration in a biosensor, the method comprising: a)providing a biosensor comprising a multisite array of test sites inwhich the conditions for interacting with a biomolecule analyte can bevaried independently, each test site comprising a support in an aqueoussolution, the support comprising one or more electrodes or anelectromagnet, and a biomolecule interface layer having one or moreimmobilized probes thereon, wherein the biomolecule interface layer iscovering the one or more electrodes and has a thickness of 5-150 nm; b)adding an electrochemically active agent, an enzyme, an enzymesubstrate, a buffer inhibitor, or a combination thereof to the aqueoussolution; and c) reacting the electrochemically active agent, theenzyme, the enzyme substrate, or combination thereof in the aqueoussolution to produce H⁺ ion or OH− ions, or increasing the diffusion ofH⁺ ions or OH⁻ ions with the buffer inhibitor, or inhibiting theinteraction between H⁺ ions or OH⁻ ions and buffering salts with thebuffer inhibitor.
 27. A biosensor for use in detecting a biomoleculeanalyte comprising: a multisite array of test sites, each test sitecomprising: a support in an aqueous solution: wherein the aqueoussolution has added thereto an electrochemically active agent, an enzyme,an enzyme substrate, a buffer inhibitor, or a combination thereof; thesupport comprises one or more electrodes or an electromagnet, and abiomolecule interface layer having one or more immobilized probesthereon; and the biosensor is configured for: during a test,independently varying for each of the test sites a degree of biologicalinteraction that can occur with a biomolecule analyte at the respectivetest site, to obtain a collection of varied signals, the varyingincluding reacting the electrochemically active agent, the enzyme, theenzyme substrate, or combination thereof in the aqueous solution toproduce H⁺ ion or OH⁻ ions, or increasing the diffusion of H⁺ ions orOH⁻ ions with the buffer inhibitor, or inhibiting the interactionbetween H⁺ ions or OH⁻ ions and buffering salts with the bufferinhibitor; and analyzing the collection of varied signals to obtain ameasurement.
 28. The biosensor according to claim 27, wherein thesupport comprises the electromagnet.
 29. The biosensor according toclaim 27, wherein the biosensor comprises a CMOS, electrode array, orTFT based system.
 30. The biosensor according to claim 27, wherein theelectrode is an indium tin oxide, gold or silver electrode.
 31. Thebiosensor according to claim 27, wherein the support is a glass orplastic support.
 32. The biosensor according to claim 27, wherein theimmobilized probe is selected from a protein, a peptide, a nucleic acidand a cell.
 33. A method for detecting a biomolecule analyte in abiological sample, the method comprising: a) providing a biosensorcomprising a multisite array of test sites in which the conditions forinteracting with a biomolecule analyte can be varied independently, eachtest site comprising a support in an aqueous solution, the supportcomprising one or more electrodes or an electromagnet, and a biomoleculeinterface layer having one or more immobilized probes thereon; b) addingin each test site an electrochemically active agent, an enzyme, anenzyme substrate, a buffer inhibitor, or a combination thereof to theaqueous solution; c) reacting the electrochemically active agent, theenzyme, the enzyme substrate, or a combination thereof in the aqueoussolution to produce H⁺ ion or OH⁻ ions, or increasing the diffusion ofH⁺ ions or OH⁻ ions with a buffer inhibitor, or inhibiting theinteraction between H⁺ ions or OH⁻ ions and buffering salts with thebuffer inhibitor; d) adding a biological sample to each test site; ande) detecting the biomolecule analyte in each test site, wherein theamounts added in step b) and the reaction in step c) are varied betweentest sites in a subset array of test sites in order to obtain sets oftest sites in which the pH or ionic concentration near electrodesurfaces in the test sites varies.
 34. The method for detecting abiomolecule analyte in a biological sample according to claim 33,wherein amount of biological sample or the concentration of thebiological sample is varied between subset arrays of test sites.
 35. Themethod for detecting a biomolecule analyte in a biological sampleaccording to claim 33, wherein the aqueous solution comprises awater-miscible organic co-solvent selected from the groups consisting ofacetonitrile, dimethyl sulfoxide (DMSO), dimethyl formamide (DMF),N,N-dimethyl acetamide (DMAc), and mixtures thereof.